Shigella species are invasive human pathogens that cause bacillary dysentery, or shigellosis, a potentially fatal diarrheal disease. The global burden of shigellosis is estimated at more than 200 million cases per year. There is currently no effective vaccine against Shigella, and drug resistance is widespread and on the rise; thus, there is a critical need to identify novel Shigella targets for immunization and new antibiotics. One promising approach is to target metabolic processes that are important for pathogenesis, but not for survival of Shigella in the host environment. Inhibiting such pathways would be less likely to select for resistant Shigella or to disrupt the normal gut microbiota. Our work has shown that Shigella flexneri uses mixed acid fermentation to break down glycolysis intermediates during growth within host cells. This process is critical for S. flexneri pathogenesis, but is not required for growth of the bacteria, either inside or outside host cells. Mixed acid fermentation leads to the production of formate, which is excreted by S. flexneri into the host cell cytosol. Formate induces expression of S. flexneri virulence genes that are required for cell-to-cell spread. Our hypothesis is that, as the bacteria multiply, formate levels reach a threshold that can be sensed by S. flexneri as a signal to begin the process of spreading to neighboring cells, thus linking cell density with the need to move deeper into the intestinal epithelium to find new resources. The goal of this study is to investigate how formate is sensed by the bacteria, and how this signal leads to changes in gene expression that promote cell-to-cell spread and evasion of host immunity. We propose to identify key players in the formate sensing pathway in order to derive a model for how the formate signal is relayed from the cell surface to its target genes. We will also determine the downstream effects of formate signaling on both S. flexneri and host cell gene expression. Historically, Shigella studies have been hampered by the lack of a physiologically relevant host-pathogen model system. We have recently demonstrated that critical aspects of Shigella pathogenesis are faithfully reproduced in human intestinal enteroids (HIEs), ?mini-intestines? derived from human intestinal biopsies. We propose to use HIEs to determine both the Shigella and host cell transcriptomes in response to formate. This will allow investigation of gene expression and metabolism during the course of a Shigella infection in fully differentiated, non-transformed, native human tissue. We have assembled the necessary strains and reagents, and we have the expertise to carry out these experiments, which we predict will lead to vital new information in the search for novel shigellosis treatment and prevention strategies.